Calcium supplementation educes sporadic colorectal adenoma recurrence, and higher serum vitamin D levels are associated with reduced risk for colorectal cancer, the second leading cause of cancer deaths in the U.S. Despite strong biological plausibility and supportive observational data, the independent and synergistic anti- neoplastic effects of calcium and vitamin D in humans are not clear. Proposed mechanisms have included protection of the colonic epithelium against bile acids, direct effects on the cell cycle, modulation of growth factors signaling, inflammation, and immune function. Based on basic science and limited human evidence, we propose to test a novel hypothesis that calcium and vitamin D supplementation strengthens the gut mucosal barrier as indicated by modulation of cell cycle, cell adhesion, receptors responsive to bacterial components, and markers of intestinal inflammation in humans. We also hypothesize that these changes in the colon will be accompanied by decreases in circulating levels of biomarkers of colonic hyperpermeability and pro-inflammatory cytokines. We will investigate this in an adjunct study to an ongoing multi-center, randomized, double-blind, placebo-controlled, 2 x 2 factorial chemoprevention clinical trial (n = 2,259) of supplemental calcium (1,200 mg elemental calcium daily as calcium carbonate) and vitamin D3 (1,000 IU daily), alone and in combination vs. placebo over 3 - 5 years (the 'parent study'). A sub-set of participants (n = 112) with non-prep biopsies of normal-appearing rectal mucosa taken at baseline and 1-year follow-up sigmoidoscopies is selected for this adjunct study. The primary aims of the proposed study are to 1) obtain preliminary data on the separate and joint effects of calcium and vitamin D3 supplementation on biomarkers of gut barrier function (Ki-67/MIB-1, caspase-3, TLR4, TLR5, ZO-1, claudin-1, occludin, mucin-17, E-cadherin, and beta-catenin) and inflammation (NFkappaB and TNFalpha) in biopsies of normal-appearing rectal mucosa, and 2) investigate whether the findings for the tissue-specific biomarkers are correlated with circulating biomarkers of gut barrier function (specific IgG and IgA to lipopolysaccharide [LPS] and flagellin, LPS binding protein [LBP], and intestinal fatty acid binding protein [I-FABP]) and inflammation (GM-CSF, IFNgamma, IL-6, IL-1beta, IL-2, IL-8, IL- 10, IL-12p70, and TNFalpha). The proposed scope of work is limited to laboratory and statistical analyses using biological samples and questionnaire data from the 'parent study'. This adjunct study offers a unique, cost-effective opportunity in a large, ongoing trial in humans to explore a novel hypothesis that calcium and vitamin D beneficially modulate gut barrier function, and to elucidate the combined effects of calcium and vitamin D on biomarkers of gut barrier function and inflammation. Understanding the role of vitamin D and calcium in gut barrier function is of great importance, not only to colorectal neoplasm chemoprevention, but also to the prevention of multiple pathological conditions of the gastrointestinal tract.